Project Summary Activated macrophages play an important role in many inflammatory diseases including septic shock and atherosclerosis. However, the molecular mechanisms limiting macrophage inflammation are not completely understood. MCP-induced protein 1 (MCPIP1) is a recently identified CCCH-zinc finger containing protein, which was significantly induced by monocyte chemotactic protein 1 (MCP-1) and thus designated as MCPIP1. In our previous works, we have identified MCPIP1 as a novel negative regulator of macrophage inflammatory activation. In our recent studies, we have further found that MCPIP1 acts as a deubiquitinating enzyme that may negatively regulates NF-B signaling by removing ubiquitin moieties from critical proteins, such as TNF receptor adaptor factors (TRAFs), RIP and IB. Consistently, MCPIP1- deficient mice spontaneously developed inflammatory syndrome and died prematurely. Macrophages from MCPIP1-/- mice showed high up-regulation of inflammatory gene expression, together with a greatly increased NF-B activation, as well as increased polyubiquitination of TRAF2 and TRAF6. Furthermore, in vitro assay directly demonstrated the deubiquitinating activity of purified MCPIP1. Based on these intriguing findings, we hypothesize that MCPIP1 represses NF-kB signaling and microphage activation mainly through deubiquitination of TRAF family. The overall objective of this proposal is to test the central hypothesis by using combined biochemical and genetic approaches. Specially, we will establish MCPIP1- mediated deubiquitination of TRAFs as an essential mechanism in the regulation of NF-B signaling as well as macrophage activation both in vitro and in vivo. We will attain the objective of this aim by using following approaches: 1) determine the direct molecular targets of MCPIP1 deubiquitinase in vitro; 2) determine the in vivo targets of MCPIP1 deubiquitinase using the primary cells and tissues from MCPIP1 knockout mouse; 3) map the active domain of MCPIP1 deubiquitinase through serial mutagenesis analysis; 4) define the functional relationship between deubiquitinase domain and RNase domain; 5) determine the relative contribution of the deubiquitinase activity of MCPIP1 to the suppression of NF-B signaling as well as macrophage activation; 6) determine the role of MCPIP1 deubiquitinase in human macrophages. Completion of the proposed studies will not only help to understand the molecular basis of macrophage activation, but also implicate in the development of novel drug therapies against inflammatory diseases such as atherosclerosis.